Negative strand RNA viral vector having autonomous replication capability

ABSTRACT

A method for reconstituting Sendai viral particles by transfecting Sendai virus to a host expressing all genes for the initial replication has been developed, enabling the Production of negative strand RNA vectors highly useful for practical applications.

This application is a continuation of PCT International Application No.PCT/JP96/03068, filed Oct. 22, 1996, which, in turn, claims the benefitof Japanese Patent Application No. 7/308,315, filed Oct. 31, 1995.

FIELD OF THE INVENTION

The present invention relates to a viral vector for the gene therapy.More specifically, this invention relates to a negative strand RNA viralvector.

BACKGROUND OF THE INVENTION

As to the gene therapy for humans and animals, therapeutic effectivenessand safety are very important factors. Especially, therapy performed byusing “viral vector” expressing a foreign gene of concern which isobtained by gene recombination of the viral genome and the foreign geneneeds to be very cautiously carried out, when such undeniablepossibilities exist as that the recombinant virus may be inserted tounspecified sites of chromosomal DNA, that the recombinant virus andpathogenic virus may be released to the natural environment, and thatthe expression level of gene transfected into cells cannot becontrolled, or the like, even though its therapeutic effectiveness isrecognized.

These days, a great number of gene therapies using recombinant virusesare performed, and many clinical protocols of gene therapy are proposed.Characteristics of these recombinant viral vectors largely depend onthose of the viruses from which said vectors are derived. The basicprinciple of viral vector is a method for transferring the desired geneinto targeted cells by utilizing the viral infectivity. By “infectivity”in this specification is meant the “capability of a virus to transferits nucleic acid, etc. into cells through its adhesiveness to cells andpenetrating capability into cells via various mechanisms includingfusion of the viral membrane and host cellular membrane”. With thesurface of recombinant viral vectors genetically manipulated to insert adesired gene are associated the nucleocapsid and envelope proteins, etc.which are derived from the parental virus and confer infectivity on therecombinant virus. These proteins enable the transfer of the enclosedrecombinant gene into cells. Such recombinant viral vectors can be usedfor the purpose of not only gene therapy, but also production of cellsexpressing a desired gene as well as transgenic animals.

Viral vectors are classified into three classes comprising theretroviral vector, DNA viral vector and RNA viral vector.

These days, the vectors most frequently used in gene therapy areretroviral vectors derived from retroviruses. Retroviruses replicatethrough the following processes. First, upon penetration into cells,they generate complementary DNAs (cDNAs) using their own reversetranscriptase as at least part of catalysts and their own RNA templates.After several steps, said cDNAs are incorporated into host chromosomalDNAs, becoming the proviruses. Proviruses are transcribed by theDNA-dependent RNA polymerase derived from the host, generating viralRNAs, which is packaged by the gene products (proteins) translated fromthe RNAs. The RNAs and proteins finally assemble to form mature virusparticles.

In general, retroviral vectors used in gene therapy, etc. are capable ofcarrying out processes up to provirus generation. However, they aredeficient viruses deprived of genes necessary for their packaging of theprogeny genome RNA so that they do not form viral particles fromprovirus. Retroviruses are exemplified by, for example, mouse leukemiavirus, feline leukemia virus, baboon type C oncovirus, humanimmunodeficiency virus, adult T cell leukemia virus, etc. Furthermore,recombinant retroviral vectors hitherto reported include those derivedfrom mouse leukemia virus [see Virology, 65, 1202 (1991), Biotechniques,9, 980 (1989), Nucleic Acids Research, 18, 3587 (1990), Molecular andCellular Biology, 7, 887 (1987), Proceedings of National Academy ofSciences of United States of America, 90, 3539 (1993), Proceedings ofNational Academy of Sciences of United States of America, 86, 3519(1989), etc.] and those derived from human immunodeficiency virus [seeJournal of Clinical Investigation, 88, 1043 (1991)], etc.

Retroviral vectors are produced aiming at efficiently inserting adesired specific DNA into the cellular chromosomal DNA. However, sincethe insertion position of the DNA is unpredictable, there is undeniablepossibilities such as the damage of normal genes, activation of oncogeneand excessive or suppressive expression of desired gene, depending theposition of insertion. In order to solve these problems, a transientexpression system using DNA viral vectors which can be used asextrachromosomal genes has been developed.

DNA viral vectors are derived from DNA viruses, having DNA as geneticinformation within viral particles. Replication of said DNA is carriedout by repeating the process of generating complementary DNA strandusing DNA-dependent DNA replicase derived from host as at least one ofcatalysts with its own DNA as template. The actual gene therapy usingadenoviral vector, a DNA viral vector usable as extrachromosomal gene,is exemplified by the article in [Nature Genetics, 3, 1-2 (1993)].However, since, in the case of DNA viral vectors, the occurrence oftheir undesirable recombination with chromosomal DNA within nucleus isalso highly possible, they should be very carefully applied as vectorsfor gene therapy.

Recently, RNA viral vectors based on RNA viruses have been developed asconceivably more safer vectors than DNA and retroviral vectors describedabove. RNA viruses replicate by repeating the processes for generatingcomplementary strands using their own RNA-dependent RNA replicase as thecatalyst with their own RNA as template.

The genome RNA of positive strand RNA viruses have dual functions as themessenger RNA (hereafter simply called mRNA), which generate proteins,depending on the translational functions of host cells, necessary forthe replication and viral particle formation and as the template forgenome replication. In other words, the genome RNA itself of positivestrand RNA viruses has a disseminative capability. In the presentspecification, by “disseminative capability” is meant “the capability toform infectious particles or their equivalent complexes and disseminatethem to other cells following the transfer of nucleic acid into hostcells by infection or artificial techniques and the intracellularreplication of said nucleic acid”. Sindbis virus classified to positivestrand RNA viruses and Sendai virus classified to negative strand RNAviruses have both infectivity and disseminative capability.Adeno-satellite virus classified in Parboviruses is infectious but notdisseminative (mixed infection with adenovirus is required for theformation of viral particles.). Furthermore, the positive strand RNAderived from Sindbis virus which is artificially transcribed in vitro isdisseminative (forming infectious viral particles when transfected intocells), but neither positive nor negative RNA strands of Sendai virusartificially transcribed in vitro is disseminative, generating noinfectious viral particles when transfected into cells.

In view of the advantage that the genome RNA functions as mRNA at thesame time, the development of RNA viral vectors derived from positivestrand RNA viruses preceded [see Bio/Technology, 11, 916-920 (1993),Nucleic Acids Research, 23, 1495-1501 (1995), Human Gene Therapy, 6,1161-1167 (1995), Methods in Cell Biology, 43, 43-53 (1994), Methods inCell Biology, 43, 55-78 (1994)]. For example, RNA viral vectors derivedfrom Semliki forest virus (SFV) [Bio/Technology, 11, 916-920 (1993)] andSindbis virus are basically of the RNA structure wherein the structuralprotein gene regions related to the viral structure are deleted, and agroup of genes encoding proteins necessary for viral transcription andreplication are retained with a desired foreign gene being linkeddownstream of the transcription promotor. Direct transfer of suchrecombinant RNA or cDNA which can transcribe said RNA [Nucleic AcidsResearch, 23, 1495-1501 (1995)] into cells by microinjection, etc.allows autonomous replication of RNA vectors containing the foreigngene, and the transcription of foreign gene inserted downstream of thetranscription promotor, resulting in the expression of the desiredproducts from the foreign gene within cells. Furthermore, the presentinventors succeeded in forming an infectious but not disseminativecomplex by the co-presence of cDNA unit (helper) for expressing theviral structural gene and that for expressing said RNA vector in thepackaging cells.

Positive strand RNA viral vectors are expected to be useful as RNAvectors with autonomous replicating capability, but their use as vectorsfor gene therapy poses the following problems.

1. Since they are of the icosohedral structure, the size of foreign geneallowed to be inserted is limited to 3,700 nucleotides at most.

2. Until nucleic acids are released from the packaged complex into thecell and replicated, as many as five processes are required, includingcellular adhesion, endocytosis, membrane fusion, decapsidation andtranslation of replication enzymes.

3. A possible formation of disseminative viral particles even in aminute quantity during packaging cannot be denied. Especially, even withattenuated viral particles, the inside RNA itself has disseminativepotency and may belatedly be amplified, making it difficult to check.

4. Since these vectors are derived from viruses transmitted to animalsby insects such as mosquitoes, when animals and humans to which suchvector genes are transferred and are mix-infected with wild typeviruses, disseminative recombinants may be formed, possibly furthercreating a risk of said recombinants being scattered to the naturalenvironment by insects.

Such problems described above are conceived to be basically overcome ifRNA viral vectors derived from negative strand RNA viruses areconstructed. That is, since negative strand RNA viruses do not have thecapsid of icosohedral structure but have a helical nucleocapsid, andalso since the envelope size of particles is known to vary depending onthe inside RNA content, they are supposed to be much less restricted,compared with positive strand RNA viruses, with respect to the size offoreign genes to be inserted when used as RNA viral vectors. Further,since a group of proteins required for transcription and replication arepackaged into particles, only two processes are required, includingcellular adhesion and membrane fusion, until nucleic acids arereplicated. Furthermore, viral RNA alone is not disseminative. Inaddition, most of negative strand RNA viruses are not transmitted byinsects.

In spite of many advantages of negative strand RNA viruses which may beused as the source of industrially useful viral vectors, no negativestrand RNA vectors applicable for gene therapy has become availableuntil now. This is probably due to tremendous difficulties inre-constituting viral particles via viral cDNA. Since the genemanipulation on the DNA level is required to insert foreign genes intovectors, so far as viral particles are not reconstructed from viral cDNAwith a foreign gene inserted, it is difficult to use negative strand RNAviruses as a vector. “Reconstruction of viral particles” refers to theformation of the original virus or a recombinant virus in vitro orintracellularly from artificially prepared cDNA encoding the viral RNAgenome.

As described above, it has been clearly demonstrated that, even if theviral RNA (vRNA) of negative strand RNA viruses or its complementarystrand RNA (cRNA; complementary RNA) alone is transferred into cells, noprogeny virus can be generated. This is a definitely different pointfrom the case of positive strand RNA viruses, whose RNA can initiateviral life cycle and generate progeny viruses, when transferred intocells. Although, in JP-A-Hei 4-211377, “methods for preparing cDNAcorresponding to a nonsegmented negative strand RNA viral genome andinfectious negative strand RNA virus” are described for measles virus, aparamyxovirus, the entire experiments of said document described in“EMBO. J., 9, 379-384 (1990)” were later proved to be not reproducible,so that the authors themselves had to withdrew all the article contents[ref. EMBO. J., 10, 3558 (1991)]. Therefore, it is obvious thattechniques described in JP-A-Hei 4-211377 for another paramyxovirus,Sendai virus, do not correspond to the related art of the presentinvention.

With regard to the reconstitution system for negative strand RNAviruses, there are reports on influenza virus [Annu. Rev. Microbiol.,47, 765-790 (1993); Curr. Opin. Genet. Dev., 2, 77-81 (1992)]. Influenzavirus is an eight-segmented negative strand RNA virus. According tothese literatures, a foreign gene was first inserted to a cDNAcorresponding to one of said segments, and the RNA transcribed from thecDNAs is assembled with the virus-derived NP protein and RNA polymeraseproteins to form an RNP. Then, cells are transfected with this RNP andsuperinfected with an appropriate intact influenza virus. A reassortantvirus emerges, in which the corresponding segment is replaced with theengineered segment, which can be selected under apporopriate pressures.Several years later, virus-reconstitution entirely from cDNA ofnonsegmented negative strand RNA virus was reported with rabies virusbelonging to rhabdoviruses [EMBO J. 13, 4195-4202 (1994)].

However, it has been difficult to use these virus reconstitutiontechniques as such for constructing vectors for gene therapy because ofthe following problems.

1. Reconstituted viruses were identified only by the expression ofmarker gene, RT-PCR, etc. No re-constitution system for the productionof vector viruses in a satisfactory yield has been established.

2. Differing from the case of positive strand RNA viruses, in order toform complexes with infectivity but deficient in disseminative potencyas vectors for gene therapy, it is necessary to enclose factors requiredfor primary transcription and replication within the complex. Notechnique for amplifying these complexes in a large scale has beenestablished.

3. For the purpose of intracellularly providing factors necessary forviral reconstitution, cells to which cDNAs are introduced aremix-infected with helper viruses such as recombinant vaccinia virus,etc. to allow transcription of the plasmids supplying those viralprotein factors in trans. It is not easy to separate these natural typeviruses from the recomstituted viruses.

Furthermore, as one problem with regard to RNA viral vectors in general,it is conceivably necessary to beforehand provide inhibitors forreplication of RNA viral vectors which have no effects on host'sreplication and transcription, providing for the case where RNAreplicated and transcribed in large amounts exerts undesirable effectson treated humans and animals. However, no such inhibitors have beendeveloped.

SUMMARY OF THE INVENTION

Objects of the present invention are to develop negative strand RNAviral vectors for practical use, methods for efficiently preparing saidvectors, and inhibitors for the replication of said vectors.

The present inventors first attempted to reconstitute Sendai virus fromnucleic acids of said virus which is a typical nonsegmented, negativestrand RNA virus, and conceived to be industrially most useful as avector from the viewpoints of safety and convenience. First, in order toapply to the reconstitution test, various investigations were performedusing cDNA encoding a Sendai virus minigenome as experimental materials.A cDNA plasmid was constructed so that the Sendai virus protein codingregion of about 14 kb is replaced with a reporter luciferase gene andthis construct is flanked by T7 promoter and hepatitis delta virusribozyme gene. As a result, the inventors found efficient conditionsregarding weight ratios among materials to be transferred into hostcells, including minigenome cDNA, cDNAs encoding the nucleocapsidprotein (N), the large protein (L), and the phosphoprotein (P), andminimizing cytotoxicity induced by the recombinant vaccinia virus toprovide the T7RNA polymerase. The N protein encapsidate the naked viralRNA to form the RNP, which is now active as the template for both viralmRNA synthesis and viral replication. Furthermore, the present inventorsobtained full-length cDNAs corresponding to both positive and negativestrands, constructed plasmids for inducing the intracellularbiosynthesis of either positive strand RNA (antigenome or cRNA) ornegative strand RNA (genome or vRNA) of Sendai virus, and transferredsaid plasmids into host cells wherein N, P, and L proteins from therespective cotransfected plasmids were expressed. As a result, theinventors first succeeded in re-constructing Sendai virus particles fromcDNAs derived thereof.

That is, the present invention comprises the followings.

1. A complex comprising an RNA molecule derived from a specificdisseminative negative strand RNA virus and viral structural componentscontaining no nucleic acids, having the infectivity and autonomous RNAreplicating capability, but deficient in the disseminative capability.

2. The complex of description 1, wherein said specific RNA virus is anegative strand RNA virus having non-segmented genome.

3. The complex of description 2, wherein said specific RNA virus isSendai virus.

4. An RNA molecule comprising Sendai viral RNA (vRNA) or itscomplementary RNA (cRNA), wherein said RNA molecule is defective in thatat least one or more than one gene coding for each of the M, F and HNproteins are deleted or inactivated.

5. A complex comprising the RNA of description 4 and viral structuralcomponents containing no nucleic acids derived from Sendai virus, havingthe infectivity and autonomous RNA replicating capability, but deficientin the disseminative capability.

6. A DNA molecule comprising a template DNA transcribable to the RNAmolecule of description 4 in vitro or intracellularly.

7. The complex of any one of descriptions 1-3 or 5, wherein the RNAmolecule contained in said complex comprises a foreign gene.

8. The complex of descriptions 3 or 5, wherein the RNA moleculecontained in said complex comprises a foreign gene.

9. The RNA molecule of description 4 comprising a foreign gene.

10. The DNA molecule of description 6 comprising a foreign gene.

11. An inhibitor for RNA replication contained in the complex of any oneof descriptions 1-3, 5, 7 or 8 comprising an inhibitory drug for theRNA-dependent RNA replication.

12. A host whereto the complex of any one of descriptions 1-3, 5, 7 or 8can disseminate.

13. The host of description 12 comprising a group of genes related tothe infectivity of the complex of any one of descriptions 1-3, 5, 7 or 8on its chromosomes, and capable of replicating the same copies of saidcomplex when infected with it.

14. The host of descriptions 12 or 13, wherein said host is animals, orcells, tissues, or embryonated eggs derived from it.

15. The host of description 14 wherein said animal is mammalian.

16. The host of description 14 wherein said animal is avian.

17. A host comprising a group of genes related to the infectivity of thecomplex of any one of descriptions 3, 5 or 8 on its chromosomes, andcapable of replicating the same copies of said complex when infectedwith it.

18. A host comprising at least more than one gene of the M, F and HNgenes of Sendai virus or genes having functions equivalent to them onits chromosomes.

19. A host comprising the M, F, or HN gene of Sendai virus or each oftheir functionally equivalent genes on its chromosomes.

20. A host comprising the M, NP, P and L genes of Sendai virus on itschromosomes (wherein each gene may be substituted with its functionallyequivalent gene, respectively).

21. A host comprising the M, F and HN genes of Sendai virus on itschromosomes (wherein each gene may be substituted with its functionallyequivalent gene, respectively).

22. A host comprising the M, F, HN, NP, P and L genes of Sendai virus onits chromosomes (wherein each gene may be substituted with itsfunctionally equivalent gene, respectively).

23. The host of any one of descriptions 17-22, wherein said host isanimal, or cell, tissue or egg derived from it.

24. The host of description 23, wherein said animal is mammalian.

25. The host of description 23, wherein said animal is avian.

26. A kit consisting of the following three components,

a. the RNA molecule contained in the complex of any one of descriptions1-3, 5, 7 or 8, or cRNA of said RNA, or a unit capable ofbiosynthesizing said RNA or said cRNA,

b. a group of enzymes required for replicating said RNA or said cRNA, ora unit capable of biosynthesizing said group of enzymes, and

c. a group of proteins related to the infectivity of said complex, or aunit for biosynthesizing said group of proteins.

27. A kit consisting of the following three components,

a. the RNA molecule contained in the complex of any one of descriptions1-3, 5, 7 or 8, or cRNA of said RNA, or a unit capable ofbiosynthesizing said RNA or said cRNA,

b. a group of enzymes required for replicating said RNA or said cRNA, ora unit capable of biosynthesizing said group of enzymes, and

c. the host of any one of descriptions 12-25.

28. A kit consisting of the following two components,

a. the complex of any one of descriptions 1-3, 5, 7 or 8, and

b. the host of any one of descriptions 12-25.

29. A kit consisting of the following three components,

a. the RNA molecule contained in the complex of any one of descriptions3, 5 or 8, or cRNA of said RNA, or a unit capable of biosynthesizingsaid RNA or said cRNA,

b. all NP, P and L proteins of Sendai virus, or a unit forbiosynthesizing said group of proteins, and

c. a group of proteins related to the infectivity of said complex, or aunit for biosynthesizing said group of proteins.

30. A kit consisting of the following three components,

a. the RNA molecule contained in the complex of any one of descriptions3, 5 or 8, cRNA of said RNA, or a unit capable of biosynthesizing saidRNA or said cRNA,

b. all NP, P and L proteins of Sendai virus, or a unit capable ofbiosynthesizing said group of proteins, and

c. the host of any one of descriptions 17-25.

31. A kit consisting of the following two components,

a. the complex of any one of descriptions 3, 5 or 8, and

b. the host of any one of descriptions 17-25.

32. A method for producing the complex of any one of descriptions 1-3,5, 7 or 8 by introducing three components of descriptions 26a, 26b and26c into a host.

33. A method for producing the complex of any one of descriptions 1-3,5, 7 or 8 by introducing both components of descriptions 27a and 27binto the host of description 27c.

34. A method for amplifying and producing the complex of description 28aby transfecting said complex to the host of description 28b.

35. A method for producing the complex of any one of descriptions 3, 5or 8 by introducing the three components of descriptions 29a, 29b and29c into a host.

36. A method for producing the complex of any one of descriptions 3, 5or 8 by introducing both components of descriptions 30a and 30b into thehost of description 30c.

37. A method for amplifying and producing the complex of description 31aby transfecting said complex into the host of description 31b.

38. The RNA molecule of description 9 wherein a gene corresponding tothe M, F, or HN gene is deleted or inactivated.

39. The RNA molecule of description 9 wherein all the genescorresponding to the M, F and HN genes are deleted or inactivated.

40. A kit consisting of the following three components,

a. the RNA molecule of description 38,

b. the host of description 20, and

c. the host of description 19.

41. A method for producing a complex by introducing the RNA molecule ofdescription 40a into the host of description 40b, and amplifying andproducing said complex by transfecting it into the host of description40c.

42. A complex produced by the method of description 41.

43. A kit consisting of the following three components,

a. the RNA molecule of description 39,

b. the host of description 22, and

c. the host of description 21.

44. A method for producing a complex by introducing the RNA molecule ofdescription 43a into the host of description 43b, and amplifying andproducing said complex by transfecting it into the host of description43c.

45. A complex produced by the method of description 44.

46. An inhibitor for RNA replication contained in the complex of eitherdescriptions 42 or 45 comprising an inhibitory drug of the RNA-dependentRNA replication.

47. A method for preparing the foreign proteins, wherein said methodcomprises the process of introducing the complex of description 7 to ahost and the process of recovering the expressed foreign proteins.

48. A method for preparing the foreign proteins of description 47,wherein the host is a cell expressing a group of genes, from among thoserelated to the disseminative capability, which are deficient in the RNAmolecule contained in the complex of description 7.

49. A culture medium or allantoic fluid containing the expressed foreignproteins, wherein said culture medium or allantoic fluid is obtained byinoculating the complex of description 7 into a host and recovering it.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C is a schematic representation of a process for generatingcomplexes of the present invention from cDNA deficient in the M gene ofSendai virus (step A) amplifying said complexes in an M-expressing cell(step B), and replication of said complexes in a normal cell (step C).

FIG. 2 is a schematic representation of the construction of apUC18/T7(+) HVJRz.DNA.

FIG. 3 is a schematic representation of the construction of apUC18/T7(−) HVJRz.DNA.

FIG. 4 is a graphical representation showing the relationship betweenthe time after the infection of Sevgp120 into CV-1 cells and levels ofHAU and gp120 expression.

DETAILED DESCRIPTIN OF THE INVENTION

Any negative strand RNA viruses with disseminative capability may beused as materials in the present invention. Although incomplete virusessuch as defective interfering particles (DI particles) and syntheticoligonucleotide may also be used as partial materials, in general, theymust have the base sequence equivalent to that of the virus withdisseminative capability. Negative strand RNA viruses of the presentinvention include, for example, Sendai virus, Newcastle disease virus,mumps virus, measles virus, respiratory syncytial virus, rinderpestvirus of cattle and canine distemper virus of Paramyxoviridae, influenzavirus of Orthomyxoviridae, vesicular stomatitis virus and rabies virusof Rhabdoviridae.

As the negative strand viral material, recombinant negative strandviruses derived from any viruses described above and retaining thedisseminative capability may be used. For example, the recombinantnegative strand virus may be the one with the gene for theimmunogenicity inactivated or a partial region of gene altered toenhance the efficiency of RNA transcription and replication.

RNAs contained in the RNA-protein complex of the present invention canbe obtained by transcribing modified cDNAs derived from any viruses orrecombinant viruses described above in vitro or intracellularly. In RNAsthus obtained, at least one gene related to the disseminative capabilityof the original virus must be deleted or inactivated, but the generelated to the autonomous replication should not. In addition, RNAmolecules with artificial sequences, which are obtained by transcribing,in vitro or intracellularly, DNA formed by inserting the genes for theautonomous replication into cDNA having both terminus structures of thevirus genome may be also used.

As described above, in the case of Sendai virus, “the genes related toautonomous replication” refer to the NP, P and L genes, and “the generelated to the disseminative capability” refers to any one of the M, Fand HN genes. Therefore, the RNA molecule of Sendai virus Z straindeficient only in the M gene, for example, is suitable as a componentcontained in the “complex” of the present invention. Also, the RNAmolecule having all the M, F and HN genes deleted or inactivated arealso preferable as the component contained in the “complex” of thepresent invention. On the other hand, it is necessary for the genesencoding the NP, P and L proteins to be expressed from RNA. However, thesequences of these genes are not necessarily the same as those of virus,and may be modified by introducing variations, or replacing by thecorresponding gene derived from other viruses, so far as thetranscription and replication activity of the resulting RNA is similarto or higher than that of the natural one.

“Virus structural component free of nucleic acid” of the presentinvention includes, for example, virus with only its RNA removed. Assuch structural component is used the one which complements theinfectivity and autonomous replicating capability at the early stage,but not the disseminative capability. In the case of Sendai virus, thecomplex composed of its RNA with only the M gene deleted, and Sendaivirus having only its RNA deleted have the infectivity and autonomousreplicating capability, but no disseminative capability. Complex maycontain other components so long as it is provided with no disseminativecapability. For example, complex may contain adhering molecule, ligand,receptors, etc. on its envelope surface for facilitating the adherenceto specific cells.

The RNA molecule contained in the complex can have an inserted foreigngene at its appropriate site. In order to express a desired protein, theforeign gene encoding said protein is inserted. In the case of Sendaiviral RNA, a sequence of bases of 6 multiplication in number ispreferably inserted between sequences R1 (5′-AGGGTCAAAGT-3′) (SEQ IDNO:5) and R2 (5′-GTAAGAAAAA-3′)(SEQ ID NO:6) [Journal of Virology, Vol.67, No. 8. (1993), p. 4822-4830]. Levels of expression of the foreigngene inserted into RNA can be regulated by virtue of the site of geneinsertion and the base sequence flanking the inserted foreign gene. Forexample, in the case of Sendai viral RNA, it is thought that there areincreasing levels of expression of the inserted gene with decreasingdistance of said gene from the 3′ terminal promoter, whose length hasnot been precisely defined yet. Preferred host cells for theintroduction of the complex to express in high quantities desiredproteins are those expressing genes deleted in the RNA molecule composedof said complex. For this, transgenic avian eggs expressing said genesare most preferable for preparing proteins in large quantities becausesaid genes complement the defects of the virus, facilitating the virusproduction and thus yielding the inserted gene product in highquantities in the allantoic fluid. Also, proteins thus expressed can berecovered from the culture medium when the avian cells are cultured invitro. In Examples 5 and 6 is used a disseminative complex in place ofnon-disseminative complex of the present invention. However, it will beclear to those skilled in the art that similar results are obtained withthe complex of the present invention as with the disseminative complexin these examples when “cells expressing genes deleted from among genesfor disseminative capability in the RNA molecule contained in thecomplex” are used as host cells.

The present inventers have confirmed that, for the efficientreconstitution of Sendai virus particles, cDNA to be introduced intocells must be in the circular form rather than in the linear form, and,for viral particle formation at a high efficiency, the transcription ofthe positive strand RNA is preferred to that of the negative strand RNAwithin cells. Although these conditions may not necessarily beapplicable to the reconstitution of all other negative strand RNAviruses, it is possible to search for appropriate conditions for thereconstitution of other negative strand RNA viruses based on thedisclosure of the present invention and conventional technology,indicating a possibility for establishing techniques to produce basicmaterials of desired negative strand viral vectors, that is, the viralreconstitution systems.

Sendai virus reconstitution can be initiated following transfection withfull-length viral RNA, either negative or positive sense, that has beensynthesized in vitro from the cDNAs. This indicates that, if cells whichexpress all viral proteins (N, P, and L) required for initialtranscription, replication, and encapsidation are constituted, therecombinant Sendai virus, eventually complexes described above can beformed entirely without using helper viruses such as vaccinia virus.Since cells which express all the three viral proteins required forinitial transcription, replication, and encapsication were alreadydescribed [J. Virology, 68, 8413-8417 (1994)], those skilled in the artmay form such complementing cells. The cell type described in saidreference are the one derived from the 293 cell line which carries threeof Sendai virus genes, namely NP, P, and L, on its chromosome,expressing proteins encoded by the three genes, NP, P, and L.

From numerous examples of viral vectors, if viral particles can beefficiently reconstructed from nucleic acids, it is obvious that thoseskilled in the art are able to readily exchange a desired viral gene,insert a foreign gene, or inactivate or delete a desired gene. Forexample, an article on the use of DI (defective interfering) particles[J. Virol., 68, 8413-8417 (1994)] clearly indicates that, when Sendaivirus RNA devoid of most of the protein-conding region but intact in itspromoter sequences at both termini can be replicated in cells, if groupof enzymes (L and P proteins) necessary for transcription andreplication and the structural protein N to encapsidate the viral RNAare provided simultaneously in the cells. Therefore, once an RNAmolecule containing a foreign gene transcribed from “specific viral cDNAdeficient in at least a part of structural genes but normal in genescoding for N, P, and L, begins to be replicated by N, P, and Lcoexpressed by the cotransfected plasmid cDNAs, a virus particle will beformed, which is infectious to and autonomously replicating in a newcell, but deficient in the disseminative potency, and can express theforeign gene. Such complexes are extremely useful as a vector for genetherapy. That is, in the present invention, with a negative strand RNAvirus, it becomes possible to prepare complexes which are infectious aswell as autonomously replicative to express a foreign gene but isdeficient in the disseminative potency.

Such complexes defective in certain viral genes can be recovered andamplified from cells which express the corresponding (to the deletedgenes) structural proteins to complement the defects of the recombinantvirus genes. Taking embryonated avian eggs into consideration as themost suitable host for proliferating such a defective, recombinantSendai virus to a high titer, it is considered that transgenic avians,their eggs and cells which carry at least one or more genes out of M, Fand HN genes of Sendai virus on chromosome are suitable for amplifyingthe complexes. Methods for preparing transgenic avians have beenreported [Poultry Sci., 65, 1445-1458 (1986); BioTechnology, 12, 60-63(1994)], and those skilled in the art should appropriately producetransgenic birds carrying at least one or more genes out of M, F and HNgenes on their chromosomes.

The present invention also provides a method for preparing the complexdescribed above. In the following, cases related to Sendai virus areexemplified. Genome of Sendai virus Z is a single stranded RNAcomprising 15384 nucleotides. Its entire base sequence has beendetermined from cDNA clones prepared by using reverse transcriptase[Nucleic Acids Research, 11, 7313-7330 (1983); Nucleic Acids Research,12, 7965-7972 (1984); Nucleic Acids Research, 14, 1545-1563 (1986)].Since its genome RNA is a negative strand, a group of enzymes andproteins are required to transcribe the genome. The newly made proteins(N, P, and L) from the primary transcripts replicate and encapsidate thenascent antigenomic RNA strand. This antigenome, in turn, is replicatedto a new genome strand by the same N, P, and L proteins. At least sixproteins including NP, P, M, F, HN, and L are known as proteins encodedby the genome RNA. It has been elucidated that, of these proteins, NP, Pand L are factors essential and sufficient for replication [Journal ofVirology, 68, 8413-8417 (1994)], and M, F and HN are componentsnecessary for constructing the viral structure. Based on these facts,when a specific RNA virus from which RNA is derived is Sendai virus, itis possible to reconstruct an infectious complex by transferring both 1)cDNA transcribable to RNA and a gene encoding the RNA polymerasenecessary for transcribing said RNA within cells or 2) an RNA moleculeitself transcribed from said cDNA in vitro into cells wherein all thegenes for the autonomous replication, NP, P, and L, and a group ofgenes, out of M, F and HN genes, that had been deleted to restrict cellto cell dissemination, are expressed. In this case, all thesetransacting genes, NP, P, L, M, F, and HN, may be transiently expressedby transfecting cells with the plasmids coding for the respective genes.However, genes related to disseminative capability at least arepreferably incorporated into cellular chromosomes to be stablyexpressed.

The complex reconstituted as above can be produced to a high titer, byinfecting cells which express genes, one or some of M, F, and HN genes,which had been deleted in the recombinant virus. Transgenic avian eggsexpressing said group of genes are preferable for this purpose toproduce the complex in a large scale.

In addition, M, F, and HN genes expressed in cells and animals are notnecessarily of the wild-type Sendai virus. Any of those with functionsequivalent to those of the wild-type will be usable. That is, any genemay be used where said gene has complementarity to the function ofSendai virus gene deleted to make the virus nondeseminating. Preferablecells to be used are host cells for Sendai virus. Any cells can betheoritically used, if they are sufficiently susceptible to andpermissive for Sendai virus infection and replication, to express M, F,and/or HN genes, and complement the defect of the recombinant Sendaivirus intracellularly produced.

Hitherto only the enhancement of expression efficiency has beenemphasized with conventional RNA virus vectors, and little efforts havebeen made for developing compounds to suppress the RNA replication toprevent unfavorable results due to excessive expression.

As the “RNA replication inhibitor” of the present invention, any drugsto inhibit RNA-dependent RNA replication may be applied, and, forexample, Ribavirin, TJ13025, etc. are used. Such replication inhibitorsare effective, for example, when health deterioration is noticed withthe cellular amplification of recombinant RNA, or when thedown-regulation of intracellular expression of foreign genes derivedfrom recombinant RNA is required.

As an embodiment of the present invention, processes for reconstitutingthe complex of the present invention from cDNA with the M gene deletedof Sendai virus (steps A-B), and those for amplifying said complex(steps B-C) are shown in FIG. 1.

In the following, the present invention will be concretely describedwith reference to Examples, but not be limited to them.

EXAMPLE 1

Preparation of Sendai Virus cDNA Plasmids pUC18/T7(−) HVJRz.DNA andpUC18/T7(+) HVJRz.DNA

Plasmid pUC18/T7(−) HVJRz.DNA was constructed by inserting a DNAmolecule comprising T7 RNA polymerase promotor, Sendai virus cDNAdesigned to be transcribed to the negative strand RNA and the ribozymegene in this order into pUC18 vector. Also, plasmid pUC18/T7(+)HVJRz.DNA was constructed by inserting a DNA molecule comprising T7 RNApolymerase promotor, Sendai virus cDNA designed to be transcribed to thepositive strand RNA and the ribozyme gene in this order into pUC18vector. Constructions of pUC18/T7(−) HVJRz.DNA and pUC18/T7(+) HVJRz.DNAare shown in FIGS. 2 and 3, respectively.

EXAMPLE 2

Reconstitution Experiment of Sendai Virus from cDNA

LLC-MK2 cells (2×10⁶) trypsinized in a usual manner were placed in a60-mm diameter plastic dish, and incubated in MEM medium (MEMsupplemented with 10% FBS) (10 ml) in a 5% CO₂ atmosphere at 37° C. for24 h. After removing the medium and washing with PBS (1 ml), asuspension of recombinant vaccinia virus vTF7-3 expressing T7 polymerasein PBS (0.1 ml) was added to the cells at the multiplicity of infection(moi) of 2. The dish was gently agitated every 15 min to thoroughlyspread the viral solution for 1 h infection. After removing the viralsolution and washing with PBS (1 ml), a medium containing cDNA, whichwas prepared as follows, was added to the dish.

Nucleic acids shown in Tables 1 and 2 (containing plasmids expressingfactors required for the replication of Sendai virus, pGEM-L, pGEM-P,and pGEM-NP, were placed in a 1.5-ml sampling tube, and adjusted to atotal volume of 0.1 ml with HBS (Hepes buffered saline; 20 mM Hepes pH7.4 containing 150 mM NaCl). In those tables, (−) and (+) cDNAsrepresent plasmids pUC18/T7(−) HVJRz.DNA and pUC18/T7(+) HVJRz. DNA,respectively, and /C and /L indicate that cDNA is introduced into cellsin the circular form and linear form after digestion of those twoplasmids with restriction enzyme MluI, respectively.

On the other hand, in a polystyrene tube were placed HBS (0.07 ml),DOTAP (Boehringer Mannheim) (0.03 ml). To this tube was added thenucleic acid solution described above, and the mixture was left standingas such for 10 min. Then, to this mixture was added the cell culturemedium described above (2 ml, MEM supplemented with 10% FBS) followed bythe vaccinia virus inhibitors, rifampicin and cytosine arabinoside C(C/Ara/C), to the final concentrations of 0.1 mg/ml and 0.04 mg/ml,respectively, resulting in the preparation of the medium containing cDNAdescribed above.

The dish described above was incubated in a 5% CO₂ atmosphere at 37° C.for 40 h. The cells in the dish were harvested using a rubber policeman,transferred to an Eppendorf tube, sedimented by centrifuging at 6,000rpm for 5 min, and re-suspended in PBS (1 ml). Aliquots of this cellsuspension, as such or after diluted, were inoculated to 10-days olddeveloping embryonated chicken eggs. That is, the cell suspension wasdiluted with PBS to the cell numbers shown in Table 1, and eggsinoculated with its 0.1 to 0.5-ml aliquots were incubated at 35° C. for72 h, then at 4° C. overnight. Chorio-allantoic fluid was recovered asthe source of reconsituted virus from these eggs using a syringe with aneedle.

Hemagglutinin unit (HAU) and plaque forming unit (PFU) of the recoveredvirus solution were assayed as follows.

HAU was determined as follows. Chicken blood was centrifuged at 400× gfor 10 min and the supernatant was discarded. Precipitates thus obtainedwere suspended in 100 volumes of PBS (−), and centrifuged at 400× g for10 min to discard the supernatant. This procedure was repeated twice toprepare an 0.1% blood cell solution in PBS. Two-fold serial dilutions ofvirus solutions were prepared, and 0.05 ml each dilution to be assayedwas dispensed into each well of 96-well titer plate. The blood cellsolution (0.05 ml each) was further added to each well, gently swirledto ensure a thorough mixing, and left at 4° C. for 40 min. Thereciprocals of the highest virus dilution to cause the hemagglutinationobservable with the naked eye was taken as HAU.

PFU was assayed as follows. CV-1 cells were grown to a monolayer on a6-well culture plate. After the culture medium was discarded, a virussolution 10-fold serially diluted (0.1 ml each) was dispensed into eachwell of the culture plate to infect the cells at 37° C. for 1 h. Duringthe infection, a mixture of 2× MEM free of serum and melted 2% agar (55°C.) was prepared, and trypsin was added to the mixture to a finalconcentration of 0.0075 mg/ml. After 1 h infection and removal of thevirus solution, the culture medium mixed with agar (3 ml each) was addedto each well of the culture plate, and incubated under a 5% CO₂atmosphere at 37° C. for 3 days. Phenol red (0.1%) (0.2 ml) was added toeach well, incubated at 37° C. for 3 h, and then removed. Unstainedplaques were counted to estimate the virus titer as PFU/ml.

Table 1 shows Sendai virus template cDNAs transfected into LLC-2 cells,amounts of cDNA factors, pGEM-L, pGEM-P, and PGEM-NP, required for theRNA replication, incubation time, cell numbers inoculated to chickeneggs, HAU and PFU values recovered into the allantoic fluid.

TABLE 1 Template pGEM pGEM pGEM amount -L -P -NP Incubation Amount cDNA(μg) (μg) (μg) (μg) time (h) of cells HAU PFU (+)cDNA/C 10 4 2 4 40 1.00× 10⁵ 512 2 × 10⁹ (+)cDNA/C 10 4 2 4 40 1.00 × 10⁵ 256 9 × 10⁸ (+)cDNA/C10 4 2 4 40 1.00 × 10⁶ 256 9 × 10⁸ (+)cDNA/L 10 4 2 4 40 1.00 × 10⁵ <2<10 (+)cDNA/L 10 4 2 4 40 1.00 × 10⁵ <2 <10 (+)cDNA/L 10 4 2 4 40 1.00 ×10⁶ <2 <10 (−)cDNA/L 10 4 2 4 40 1.00 × 10⁴ <2 <10 (−)cDNA/L 10 4 2 4 401.00 × 10⁵ <2 <10 (−)cDNA/L 10 4 2 4 40 1.00 × 10⁶ <2 <10 (−)cDNA/C 10 42 4 40 1.00 × 10⁴ <2 <10 (−)cDNA/C 10 4 2 4 40 1.00 × 10⁵ <2 <10(−)cDNA/C 10 4 2 4 40 1.00 × 10⁶ 4 8 × 10³

Samples showing both HAU and PFU were sedimented byultra-centrifugation, re-suspended, purified by a sucrose densitygradient centrifugation from 20% to 60%. The viral proteins of thuspurified virions were fractionated by 12.5% SDS-PAGE. Each viral proteinrecovered from cDNAs samples was the same in size as that of theconventional Sendai virus.

These results demonstrated that Sendai virus can be reconstituted byintroducing cDNAs into cells, and that virus particles are moreefficiently reconstituted by introducing cDNAS transcribing positivestrand RNAs as compared with those transcribing negative strand RNAs,and further by introducing cDNAs in the circular form rather in thelinear form. The coexisting vaccinia virus in an amount of ca 10⁴ PFU/mlin the allantoic fluid was readily eliminated by the virus once again ineggs at a dilution of 10⁻⁷ or 10⁻⁸. This limiting dilution protocol wasused to prepare vaccinia-free stock of recovered Sendai virus in thisand all subsequent studies.

EXAMPLE 3

Survey of RNA Replication Factors Required for Sendai VirusReconstitution

Experiments were performed to examine whether all three plasmidsexpressing the L, P, and NP proteins were required for thereconstitution of Sendai virus. Experimental methods were similar tothose described in Example 2 except that any combinations of two out ofPGEM-L, pGEM-P, and PGEM-NP plasmids or only one out of them, instead ofall these three combined as in Example 2, were introduced together witha template cDNA into cells.

Table 2 shows Sendai virus template cDNAs introduced into LLC-MK2 cells,amounts of the cDNA plasmids required for RNA replication in trans,incubation time, number of cells inoculated into chicken eggs, andvalues of HAU and PFU.

TABLE 2 Template Incubation Number of amount pGEM pGEM pGEM time cellscDNA (μg) -L -P -NP (h) inoculated HAU PFU (+)cDNA/C 10 4 2 4 40 1.00 ×10⁵ 256 6 × 10⁸ (+)cDNA/C 10 4 2 4 40 1.00 × 10⁶ 512 4 × 10⁹ (+)cDNA/C10 0 2 4 40 1.00 × 10⁶ <2 <10 (+)cDNA/C 10 0 2 4 40 1.00 × 10⁶ <2 <10(+)cDNA/C 10 4 0 4 40 1.00 × 10⁶ <2 <10 (+)cDNA/C 10 4 0 4 40 1.00 × 10⁶<2 <10 (+)cDNA/C 10 4 2 0 40 1.00 × 10⁶ <2 <10 (+)cDNA/C 10 4 2 0 401.00 × 10⁶ <2 <10 (+)cDNA/C 10 0 0 4 40 1.00 × 10⁶ <2 <10 (+)cDNA 10 0 04 40 1.00 × 10⁶ <2 <10 (+)cDNA/C 10 0 2 0 40 1.00 × 10⁶ <2 <10 (+)cDNA/c10 0 2 0 40 1.00 × 10⁶ <2 <10 (+)cDNA/C 10 4 0 0 40 1.00 × 10⁶ <2 <10(+)cDNA/C 10 4 0 0 40 1.00 × 10⁶ <2 <10

As shown in Table 2, no virus reconstitution was observed by introducingany combinations of two out of these three factors into cells,confirming the necessity of all three proteins L, P, and NP for thevirus reconstitution.

EXAMPLE 4

Reconstitution Experiment of Sendai Virus in Vitro from Transcribed RNAs

Since the reconstitution of Sendai virus from the functional cDNA cloneswas described in Example 2, it was further examined whethertranscription products of said cDNAs in vitro, that is, v or (−) RNA andc or (+) RNA, can initiate and support similar reconstitution.

After the Sendai virus cDNA plasmids, pUC18/T7(−) HVJRz.DNA andpUC18/T7(+) HVJRz.DNA, were linearized with restriction enzyme MluI,using these DNAs as templates, RNA synthesis was performed in vitro witha purified T7 polymerase preparation (EPICENTRE TECHNOLOGIES:Ampliscribe T7 Transcription Kit). The method for synthesizing in vitroRNAs essentially followed the protocols provided with the kit. Using RNAproducts thus obtained in place of cDNAs in Example 2, similarexperiments were performed, and the virus production was estimated by HAtest. Results are shown in Table 3.

TABLE 3 Template pGEM- pGEM- pGEM- Number of amount L P NP Incubationcells cDNA (μg) (μg) (μg) (μg) time (h) inoculated HAU PFU in vitro 10 42 4 40 1.00 × 10⁶ 512 2 × 10⁹ (−)RNA in vitro 10 4 2 4 40 1.00 × 10⁶ 512ND (−)RNA in vitro 10 4 2 4 40 1.00 × 10⁶ 2 5 × 10³ (+)RNA in vitro 10 42 4 40 1.00 × 10⁶ <2 ND (+)RNA

These results indicate that virus can be reconstituted by introducingeither negative or positive sense strand RNAs into cells.

EXAMPLE 5

Expression of Foreign Genes Inserted into Sendai Viral Vectors in HostCells

1. Preparation of Sendai virus vector “pSeVgp120” inserted with aforeign gene, the gp120 of human immunodeficiency virus type 2 (HIV)

Using a set of primers comprising primer a

(5′-TGCGGCCGCCGTACGGTGGCAATGAGTGAAGGAGAAGT-3′) (SEQ ID NO:1) and primerd (5′-TTGCGCCCGCGATGAACTTTCACCCTAAGTTTTTTATTACTACGGCG (SEQ ID NO:2);TACGTCATCTTTTTTCTCTCTGC-3′)

the HIV-1gp120 gene was amplified on “pN1432” or a full-length cDNA ofHIV-1 strain NL43 by the standard PCR techniques. PCR products weresubjected to TA cloning, digested with NotI, and then inserted into theNotI site of “pSeV18⁺”. pSeV18⁺ contains an additional 18 nucleotidesequence with a unique NotI restriction site which is placed before theORF of NP gene of pUC/T7(+) HVJRz. Then, E. coli cells were transformedwith this recombinant plasmid. DNAs were extracted from each colony ofE. coli by the “Miniprep” method, digested with DraIII, and thenelectrophoresed. Positive clones (designated “clone 9” hereafter) wereselected by confirming to contain DNA fragments of the size expectedfrom the insertion. After DNA fragments were confirmed to have theauthentic nucleotide sequence, DNAs were purified by a cesium chloridedensity gradient centrifugation. pSeV18⁺ inserted with the gp120 gene isdesignated “pSeVgp120” hereafter.

2. Reconstitution of Sendai virus containing pSevgp120 (Sevgp120) andanalysis of gp120 expression

Reconstitution of the virus from pSevgp120 in LLCMK2 cells, the virusrecovery from allantoic fluid of embryonated chicken eggs, and assay ofthe viral HAU were done exactly as described in Example 2. The recoveredvirus was also examined for the expression of gp120 by ELISA as follows.

Samples (100 μl each allantoic fluid) were dispensed into each well of a96-well plate which had been coated with monoclonal antibody againstHIV-1, and incubated at 37° C. for 60 min. After washing with PBS,HRP-linked anti-HIV-1 antibody (100 μl each) was added to each well, andincubated at 37° C. for 60 min. After washing with PBS,tetramethylbenzidine was added to each well, and amounts of reactionproduct converted by the action of HRP under acidic conditions weredetermined by following the optical density at 450 nm to estimate theexpression amount of gp120. Results are shown in the left-hand column inTable 4.

The virus solution thus obtained was inoculated to CV-1 cells, andsimilarly examined for gp120 expression as follows. CV-1 cells weredispensed to a culture plate at 5×10⁵ cells/plate, grown, and then theculture medium was discarded. After washing with PBS (−), the viralsolution was added to the cells at the multiplicity of infection of 10,and incubated at 37° C. for 1 h. After the virus solution was discarded,washed with PBS (−), a plain MEM medium (MEM medium supplemented withantibiotics AraC and Rif, and trypsin) was added to the cells, andincubated at 37° C. for 48 h. After the reaction, the medium wasrecovered and assayed for HAU (by a similar method as described inExample 2) and examined for the expression of gp120 (by ELISA). Resultsare shown in the center column of Table 4. In addition, the supernatantof CV-1 cell culture medium was inoculated to embryonated chicken eggsagain, and the virus solution thus obtained was assayed for HAU and alsoexamined for the gp120 expression (by ELISA). Results are shown in theright hand column of Table 4.

TABLE 4 (μg/ml) Allantoic Allantoic fluid (F1) CV-1 medium (F1) fluid(F2) gp120 (HAU) gp120 (HAU) gp120 (HAU) 0.10 (4)  3.46 (128) 0.15 (32)1.81 (128) 1.56, 1.21 (512, 512) 0.05 (32) 2.20 (128)

As shown in Table 4, markedly high concentrations of gp120 were detectedin CV-1 cells in culture (center column of the Table), and also in theallantoic fluids from embryonated chicken eggs inoculated again with thevirus (right-hand column of the Table). In the left-hand and centercolumns of the Table are shown the mean values of three clones.

Furthermore, the expression of gp120 was analyzed by Western blotting.After the culture medium of CV-1 cells infected with Sevgp120 wascentrifuged at 20,000 rpm for 1 h to sediment virus, the supernatant wastreated with either TCA (10%, v/v) for 15 min on ice or 70% ethanol at−20° C., and centrifuged at 15,000 rpm for 15 min. Proteins thusprecipitated were solved in an “SDS-PAGE sample buffer” (DaiichiChemicals) at 90° C. for 3 min, and then subjected to electrophoresis on10% SDS-polyacrylamide gel (SDS-PAGE). Proteins thus fractionated weretransferred to PVDF membranes (Daiichi Chemicals), reacted withmonoclonal antibody 902 at room temperature for 1 h, and then washedwith T-TBS. The membranes were reacted with anti-mIgG (Amersham) at roomtemperature for 1 h, and washed with T-TBS. The membranes were thenreacted with HRP-linked protein A (Amersham) at room temperature for 1h, washed with T-TBS, and 4-chloro-1-naphthol (4CNPlus) (DaiichiChemicals) was added to detect gp120. As a result, protein bands werevisualized at positions corresponding to the expected molecular weightof gp120.

In addition, effects of postinfection time of CV-1 cells transfectedwith SeVgp120 on the HAU value and gp120 expression amount wereanalyzed. CV-1 cells (5×10⁶) dispensed to 10-cm plate were infected withSevgp120 at the multiplicity of infection of 10, and the culture medium(1 ml each) was postinfectionally recovered at 30, 43, 53 and 70 h,mixed with an equal volume of the fresh medium, and subjected to HAUassay, gp120 expression examination (by ELISA) and Western blotting.Results are shown in FIG. 4. As clearly shown in FIG. 3, the productionof gp120 tends to increase with the increasing HA titer of Sendai virus.

EXAMPLE 6

Analyses of Sevgp120 Propagation and gp120 Production in Various Typesof Cells

Using similar methods as those in Example 5 except for the use ofvarious types of cells, HAU and gp120 expression levels (by ELISA) wereassayed. Results are shown in Table

TABLE 5 Cell type Hours (postinfection) HAU rgp120 (μg/ml) CV-1 96 322.5 LLCMK2 48 16 0.5 CHO 55  4 0.46 NIH3T3 48  4 0.25 MT4 24 16 0.8MOLT4/ 24 16 1.2

In the left-hand column of the Table are shown the postinfection times(hours) of various types of cells transfected with SeVgp120. As aresult, SeVgp120 propagation and gp120 expression were detected in alltypes of cells tested.

EXAMPLE 7

Studies on the Expression of Luciferase Gene Inserted into the SendaiViral Vector in Host Cells

In order to isolate the luciferase gene for inserting to vectors, theluciferase gene bounded by the engineered NotI sites on both termini wasconstructed by the standard PCR using a set of primers

[5′-AAGCGGCCGCCAAAGTTCACGATGGAAGAC-3′) (30mer) (SEQ ID NO: 3)] and[5′-TGCGGCCGCGATGAACTTTCACCC- (69mer) (SEQ ID NO: 4)TAAGTTTTTCTTACTACGGATTATTACAATTTGGACTTTCCGCCC-3′

with the minigenome encoding plasmid, “pHvluciRT4”, as a template. ThePCR product was cloned into the NotI window of pSeV18⁺ to obtain arecombinant Sendai virus vector to which the luciferase gene isinserted. Then, this recombinant vector was transfected into LLCMK2cells, and after 3 cycles of freezing and thawing, the cells wereinoculated into embryonated chicken eggs. Chorio-allantoic membranes ofdeveloping eggs were excised out, twice washed with cold PBS (−), and,after the addition of lysis buffer (Picagene WAKO) (25 μl) and thoroughmixing, centrifuged at 15,000 rpm for 2 min. To the supernatant (5 μleach) was added the substrate (IATRON) (50 μl), and the mixture wasdispensed into each well of a 96-well plate. Fluorescent intensity wasmeasured with a luminometer (Luminous CT-9000D, DIA-IATRON), and theenzyme activity was expressed as counts per second (CPS). As a result,an extremely high luciferase activity was detected. The egg grownrecombinant virus was purified by passaging once again in eggs, so thatthe stock virus did not contain helper vaccinia virus. This stock viruswas then used to infect CV-1 cells and examine luciferase expression inthese cells. As shown in Table 6, again, extremely high luciferaseactivity was detected for infected CV-1 cells at 24-h postinfection(Table 6). In these experiments, Sendai virus which did not carry theluciferase gene was used as control (represented by “SeV” in the table).Results obtained from two clones are shown in the table.

TABLE 6 Fluorescence intensity (counts/10 sec) Chorio-allantoic membraneCV-1 (24 h postinfection) Luc/SeV  669187 2891560 8707815 SeV    69   48    23    49

In the present invention, a system has been established allowing theefficient rescue of viral particles from cDNAs of negative strandviruses, and also a method has been developed enabling the productionand amplification of “complexes comprised of RNAs derived fromdisseminative specific negative strand RNA virus and viral structuralcomponents containing no nucleic acids so as to have the infectivity andautonomous RNA replicating capability but no disseminative potency”.Since said complexes can replicate only within infected cells but notspread from cell to cell, these techniques are especially useful in thefields of gene therapy, etc. wherein therapeutical safety is highlyappreciated.

4 38 base pairs nucleic acid single linear Other 1 TGCGGCCGCC GTACGGTGGCAATGAGTGAA GGAGAAGT 38 69 base pairs nucleic acid single linear Other 2TTGCGGCCGC GATGAACTTT CACCCTAAGT TTTTVTTACT ACGGCGTACG TCATCTTTTT 60TCTCTCTGC 69 30 base pairs nucleic acid single linear Other 3 AAGCGGCCGCCAAAGTTCAC GATGGAAGAC 30 68 base pairs nucleic acid single linear Other4 TGCGGCCGCG ATGAACTTTC ACCTAAGTTT TTCTTACTAC GGATTATTAC AATTTGGACT 60TTCCGCCC 68

What is claimed is:
 1. A complex devoid of infectious helper virus,wherein said complex is produced by introducing into a cell: a Sendaivirus RNA molecule comprising a foreign gene; wherein said RNA moleculeis selected from a group consisting of an RNA molecule and a cRNA ofsaid RNA molecule; wherein said RNA molecule has at least one geneassociated with the disseminative capacity of Sendai virus deleted orinactivated such that the RNA molecule is rendered non-disseminative andwherein said at least one gene is selected from the list of M, F and HN;and Wherein said cell expresses viral structural components comprisingSendai viral proteins essential for autonomous replication of Sendaivirus, wherein said Sendai viral proteins provide the protein encoded bysaid at least one gene deleted or inactivated in the RNA molecule so asto provide the complex with cell infectivity and autonomous replicatingcapabilities but without the ability to disseminate.
 2. The complex ofclaim 1, wherein said Sendai viral proteins comprise NP and RNApolymerase proteins derived or isolated from Sendai virus.
 3. Thecomplex of claim 1, wherein said viral proteins comprise Sendai viralproteins NP, P. and L.
 4. A packaging cell to which the complex of claim1 can disseminate, wherein said packaging cell is a mammalian or aviancell wherein said packaging cell comprises the one or more genesassociated with the disseminative capacity of the Sendai virus deletedor inactivated in said RNA molecule of said complex, wherein saidpackaging cell produces a dissemination deficient yet replication andinfection competent Sendai virus particle comprising the foreign genewhen said complex is introduced into said packaging cell.
 5. Thepackaging cell of claim 4, wherein said packaging cell is selected fromthe group consisting of: i. a cell comprising at least one gene selectedfrom the group consisting of Sendai virus genes M, F, and HN; ii. a cellcomprising a Sendai virus M gene; iii. a cell comprising a Sendai virusgenes M, NP, P, and L; and iv. a cell comprising a Sendai virus genes M,F, HN, NP, P. and L.
 6. The packaging cell of claim 4, wherein saidpackaging cell is derived or isolated from an avian egg.
 7. Thepackaging cell of claim 4, wherein said packaging cell is derived orisolated from a mammalian or avian cell.
 8. A method for preparing aforeign protein, wherein said method comprises the steps of introducingthe complex of claim 1 into the packaging cell of claim 4 and recoveringthe protein encoded by the foreign gene comprised in said RNA molecule.9. A method for producing a dissemination deficient yet replication andinfection competent Sendai viral particle comprising a foreign gene,wherein said method comprises the steps of introducing the complex ofclaim 1 into the packaging cell of claim
 4. 10. The packaging cell ofclaim 4, wherein said packaging cell does not express a heterologous RNApolymerase.
 11. The method of claim 9, wherein said packaging cell doesnot express a heterologous RNA polymerase.